627. Bioinformatic Approach to Design a Plasmodium falciparum PfRipr Multi-Epitope Vaccine Construct

Abstract Background Malaria infections remain an enormous contributor to global fatalities with an estimated death toll of 619,000 in 2021 and 77% of deaths being children aged under 5 years. Transmitted via mosquito, Plasmodium falciparum is the most lethal parasite of its genus but has evaded many treatment and vaccine efforts due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise for malaria vaccine development due to their ease of development and ability to target dominant regions in antigenically variable pathogens. The recently characterized protein P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. Merozoite Invasion Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. Methods Using P. falciparum sequences collected from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes with regard to T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. Putative epitopes were filtered using allele coverage, conservation, antigenicity, and allergenicity. B-Cell Epitope Prediction Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction. Epitope Sorting and Filtering Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope). Results Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. These epitopes were used to design a multi-epitope-based bloodstage vaccine construct against P. falciparum. MHC Epitopes Partial list of MHC epitopes found in Burkina Faso and Uganda datasets. B-Cell Epitopes B-cell epitopes found in Burkina Faso and Uganda datasets. PfRipr 3D Structure with B-cell Epitopes 3D model of PfRipr predicted via AlphaFold with labeled B-cell epitopes Conclusion To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to P. falciparum sequence datasets collected from other malaria endemic regions to develop vaccines effective against circulating strains. Multi-Epitope Vaccine Construct Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Disclosures All Authors: No reported disclosures

Background.Malaria infections remain an enormous contributor to global fatalities with an estimated death toll of 619,000 in 2021 and 77% of deaths being children aged under 5 years.Transmitted via mosquito, Plasmodium falciparum is the most lethal parasite of its genus but has evaded many treatment and vaccine efforts due to its complex life cycle and redundant invasion mechanisms.Epitope-based vaccines hold significant promise for malaria vaccine development due to their ease of development and ability to target dominant regions in antigenically variable pathogens.The recently characterized protein P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine.

Merozoite Invasion
Methods.Using P. falciparum sequences collected from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes with regard to T-cell receptor binding and B-cell recognition.T-cell receptor binding was predicted using NetMHCpan searching against MHC I and II alleles with high regional frequencies.Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes.Putative epitopes were filtered using allele coverage, conservation, antigenicity, and allergenicity.

B-Cell Epitope Prediction
Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction.
Epitope Sorting and Filtering Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope).
Results.Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous.These epitopes were used to design a multi-epitope-based bloodstage vaccine construct against P. falciparum.To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models.The pipeline of immunoinformatic analyses formulated in this project can be further applied to P. falciparum sequence datasets collected from other malaria endemic regions to develop vaccines effective against circulating strains.
Multi-Epitope Vaccine Construct Schematic representation of hypothetical multi-epitope vaccine construct.FliC is added to the N-terminus to stimulate and amplify immune response.The EAAAK linker is used to separate the bifunctional fusion protein domains.AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding.GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity.A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes.
Disclosures.All Authors: No reported disclosures 1 Université de Sherbrooke, Sherbrooke, Quebec, Canada 2 Université de Sherbrooke/ CIUSSSE-CHUS, Sherbrooke, Quebec, Canada 3 Universite de Sherbrooke, Sherbrooke, QC, Canada 4 CIUSSS NIM -HSCM, Montreal, Quebec, Canada MHC Epitopes Partial list of MHC epitopes found in Burkina Faso and Uganda datasets.B-Cell Epitopes B-cell epitopes found in Burkina Faso and Uganda datasets.PfRipr 3D Structure with B-cell Epitopes 3D model of PfRipr predicted via AlphaFold with labeled B-cell epitopes Conclusion.